The eukaryotic heat shock protein 90s (HSP90s) are ubiquitous chaperone proteins, which bind and hydrolyze ATP. The HSP90 family of proteins includes four known members: Hsp90 α and β, Grp94 and Trap-1. The roles of HSP90s in cellular functions are not completely understood, but recent studies indicate that HSP90s are involved in folding, activation and assembly of a wide range of proteins, including key proteins involved in signal transduction, cell cycle control and transcriptional regulation. For example, researchers have reported that HSP90 chaperone proteins are associated with important signaling proteins, such as steroid hormone receptors and protein kinases, including many implicated in tumorigenesis, such as Raf-1, EGFR, v-Src family kinases, Cdk4, and ErbB-2 (Buchner J., 1999, TIBS, 24:136-141; Stepanova, L. et al., 1996, Genes Dev. 10:1491-502; Dai, K. et al., 1996, J. Biol. Chem. 271:22030-4).
In vivo and in vitro studies indicate that without the aid of co-chaperones HSP90 is unable to fold or activate proteins. For steroid receptor conformation and association in vitro, HSP90 requires Hsp70 and p60/Hop/Sti1 (Caplan, A., 1999, Trends in Cell Biol., 9: 262-68). In vivo HSP90 may interact with HSP70 and its co-chaperones. Other co-chaperones associated with HSP90s in higher eukaryotes include Hip, Bag1, HSP40/Hdj2/Hsj1, Immunophillinis, p23, and p50 (Caplan, A. supra).
Ansamycin antibiotics are natural products derived from Streptomyces hygroscopicus that have profound effects on eukaryotic cells. Many ansamycins, such as herbimycin A (HA) and geldanamycin (GM), bind tightly to a pocket in the HSP90 (Stebbins, C. et al., 1997, Cell, 89:239-250). The binding of ansamycins to HSP90 has been reported to inhibit protein refolding and to cause the proteasome dependent degradation of a select group of cellular proteins (Sepp-Lorenzino, L., et al., 1995, J. Biol. Chem., 270:16580-16587; Whitesell, L. et al., 1994, Proc. Natl. Acad. Sci. USA, 91: 8324-8328).
The ansamycins were originally isolated on the basis of their ability to revert v-src transformed fibroblasts (Uehara, Y. et al., 1985, J. Cancer Res., 76: 672-675). Subsequently, they were said to have antiproliferative effects on cells transformed with a number of oncogenes, particularly those encoding tyrosine kinases (Uehara, Y., et al., 1988, Virology, 164: 294-98). Inhibition of cell growth is associated with apoptosis and, in certain cellular systems, with induction of differentiation (Vasilevskaya, A. et al., 1999, Cancer Res., 59: 3935-40). A GM derivative is currently in phase I clinical trials.
The use of ansamycins as anticancer agents are described in U.S. Pat. Nos. 4,261,989, 5,387,584 and 5,932,566. The preparation of the ansamycin, geldanamiycin, is described in U.S. Pat. No. 3,595,955 (incorporated herein by reference).
The ansamycin-binding pocket in the N-terminus of Hsp90 is highly conserved and has weak homology to the ATP-binding site of DNA gyrase (Stebbins, C. et al., supra; Grenert, J. P. et al., 1997, J. Biol. Chem., 272:23843-50). This pocket has been reported to bind ATP and ADP with low affinity and to have weak ATPase activity (Proromou, C. et al., 1997, Cell, 90: 65-75; Panaretou, B. et al., 1998, EMBO J., 17: 4829-36). In vitro and in vivo studies are said to indicate that occupancy of the pocket by ansamycins alters HSP90 function and inhibits protein refolding. At high concentrations, ansamycins have been reported to prevent binding of protein substrates to HSP90 (Scheibel, T., H. et al., 1999, Proc. Natl. Acad. Sci. USA 96:1297-302; Schulte, T. W. et al., 1995, J. Biol. Chem. 270:24585-8; Whitesell, L., et al., 1994, Proc. Natl. Acad. Sci. USA 91:8324-8328). Alternatively, they have also been reported to inhibit the ATP-dependent release of chaperone-associated protein substrates (Schleider, C., L. et al., 1996, Proc. Natl. Acad. Sci. USA, 93:14536-41; Sepp-Lorenzino et al., 1995, J. Biol. Chem. 270:16580-16587). In both models, the unfolded substrates are said to be degraded by a ubiquitin-dependent process in the proteasome (Schneider, C., L., supra; Sepp-Lorenzino, supra.)
In both tumor and nontransformed cells, binding of ansamycins to HSP90 has been reported to result in the degradation of a subset of signaling regulators. These include Raf (Schulte, T. W. et al., 1997, Biochem. Biophys. Res. Commun. 239:655-9; Schulte, T. W., et al., 1995, J. Biol. Chem. 270:24585-8), nuclear steroid receptors (Segnitz, B., and U. Gehring. 1997, J. Biol. Chem. 272:18694-18701; Smith, D. F. et al., 1995, Mol. Cell. Biol. 15:6804-12), v-src (Whitesell, L., et al., 1994, Proc. Natl. Acad. Sci. USA 91:8324-8328) and certain transmembrane tyrosine kinases (Sepp-Lorenzino, L. et al., 1995, J. Biol. Chem. 270:16580-16587) such as EGF receptor (EGFR) and Her2/Neu (Hartmann, F., et al., 1997, Int. J. Cancer 70:221-9; Miller, P. et al., 1994, Cancer Res. 54:2724-2730; Mimnaugh, E. G., et al., 1996, J. Biol. Chem. 271:22796-801; Schnur, R. et al., 1995, J. Med. Chem. 38:3806-3812). The ansamycin-induced loss of these proteins is said to lead to the selective disruption of certain regulatory pathways and results in growth arrest at specific phases of the cell cycle (Muise-Heimericks, R. C. et al., 1998, J. Biol. Chem. 273:29864-72).
Cyclin D in complex with Cdk4 or Cdk6 and cyclin E-Cdk2 phosphorylate the protein product of the retinoblatoma gene, Rb. Researchers have reported that the protein product of the Rb gene is a nuclear phosphoprotein, which arrests cells during the G1 phase of the cell cycle by repressing transcription of genes involved in the G1 to S phase transition (Weinberg, R. A., 1995, Cell, 81:323-330). Dephosphorylated Rb is said to inhibit progression through late G1, in part, through its interaction with E2F transcription family members, which ultimately represses the transcription of E2F target genes (Dyson, N., 1998, Genes Dev., 12: 2245-2262). Progressive phosphorylation of Rb by the cyclin-dependent kinases in mid to late G1 leads to dissociation of Rb from Rb-E2F complexes, allowing the expression of E2F target genes and entry into the S phase.
The retinoblastoma gene product is mutated in several tumor types, such as retinoblastoma, osteosarcoma and small-cell lung cancer. Research also indicates that in many additional human cancers the function of Rb is is disrupted through neutralization by a binding protein, (e.g., the human papilloma virus-E7 protein in cervical carcinoma; Ishiji, T, 2000, J Dermatol., 27: 73-86) or deregulation of pathways ultimately responsible for its phoshorylation. Inactivation of the Rb pathway often results from pertubation of p16INK04a, Cyclin D1, and Cdk4.
The retinoblastoma gene product, besides being a target of human papilloma E7 protein, is also the target of other oncogenic viral gene products. For example, studies indicate that the simian virus 40 large T antigen inactivates the Rb family of proteins, including Rb, p107, and p130 (Zalvide, J. H. et al., 1998, Mol. Cell. Biol., 18: 1408-1415). Research also indicates that transformation by adenovirus requires E1A binding to Rb (Egan, C. et al., 1989, Oncogene, 4:383-388).
Scientists estimate that over 70 types of papilloma viruses infect humans (HPV) (Sasagawa, T. et al., 1996, Clinical Diag. Lab. Immunol, 3: 403-410). Of these several are associated with malignancies of humans, particularly cervical cancers (Bosch et al., 1995, J. Natl. Cancer Inst., 87:796-802). Recent evidence also implicates HPV in some head and neck cancers. Several types of HPV are associated with an intermediate to high risk of malignancies (types 16, 18, 31, 33, 35, 45, and 56) (Sasagawa, T., et al., supra). In infections with these HPV, the viral genome integrates into the genome of the infected cell with subsequent expression of transforming genes E6 and E7. Data indicate that the products of these genes may promote malignant transformation by altering the functions of two cellular tumor suppressor proteins (p53 and Rb). E6 causes the proteolytic degradation of p53 (Scheffiner, M. et al., 1990, Cell, 63: 1129-1136. E7 complexes with Rb causing its release from transcription factor E2F, leading to the activation of genes involved in cell proliferation (Dyson, N. et al., 1988, Science, 243: 934-937.).
Most cancer therapies are not successful with all types of cancers. For example, solid tumor types ultimately fail to respond to either radiation or chemotherapy. There remains a need for cancer treatments which target specific cancer types. The present invention satisfies these needs and provides related advantages as well. The present invention provides novel methods for treating cell proliferative disorders and viral infections associated with retinoblastoma negative or deficient cells.